INTRODUCTION
The application and interaction of pharmacological agents with circadian rhythm are gaining momentum in practice [1]. Studies on drugs and their variation with time of administration have revealed that drug efficacy and toxicity vary with time and seasons [2]. This is very important, especially for drugs with narrow therapeutic indices or drugs with known organ-specific toxicity, like gentamicin and artesunate [3,4].
Artesunate is a derivative of artemisinin used to treat the Plasmodium parasite [5]. Despite its benefits, it has hepatotoxic properties [6], mainly when combined with another pharmacological agent [7]. Gentamicin is an aminoglycoside antibiotic used in bacterial infections sensitive to gentamicin [8]. It has a hepatotoxicity profile [9], and previous studies have shown that gentamicin is nephrotoxic and ototoxic [10]. When co-administered, the combined hepatotoxic effects of artesunate and gentamicin may be synergistic and potentially modulated by temporal biological rhythms.
As a central organ for drug metabolism and detoxification, the liver exhibits significant circadian variation in the expression and activity of metabolic enzymes, notably the cytochrome P450 family [11]. These rhythmic patterns influence drug clearance and the generation of reactive metabolites that may contribute to organ toxicity [11]. Seasonal variation driven by changes in environmental cues such as light, temperature, and infection rates can also affect liver function and the risk of adverse drug reactions [12]. In malaria-endemic regions, seasonal peaks in disease incidence may coincide with increased drug exposure, potentially compounding the risk of drug-induced liver injury.
This work investigated how diurnal and circannual changes can affect the severity of artesunate-gentamicin hepatotoxicity. We examined chronobiological indices that modulate liver vulnerability, focusing on highlighting the relevance of drug timing in avoiding or reducing drug toxicity and improving therapeutic outcomes.
Artesunate
WHO recommends artesunate for severe malaria [13]. It is a water-soluble hemisuccinate derivative of artemisinin. Artesunate acts by generating reactive oxygen species (ROS), thereby inhibiting the parasite’s mitochondrial function, leading to the parasite’s death [14]. Although well tolerated, prolonged usage is associated with hepatocellular injuries [15]. Artesunate is primarily metabolized in the liver by esterases and cytochrome P450 enzymes; CYP isoforms are known to show circadian variation [16].
Gentamicin
Gentamicin is commonly used to treat gram-negative bacterial infections and is sometimes co-administered with artesunate in malaria-endemic regions to address secondary infections [17]. Primarily, gentamicin is known to have circadian-dependent nephrotoxicity [18]. However, gentamicin has also been reported to cause hepatotoxicity when used in high doses for a long time [19]. Studies suggest that it can accumulate in the liver and contribute to mitochondrial dysfunction, oxidative stress, and hepatocellular damage [19].
Circadian rhythm and hepatic metabolism
The hepatic system is a primary peripheral clock regulated by the central suprachiasmatic nucleus. Hepatic enzymes, such as CYP450 isoforms, show a diurnal variation in expression and activity that affects time-dependent changes in drug absorption, metabolism, and excretion, eventually altering therapeutic efficacy and possible toxicity [20]. Previous studies demonstrated that drug-induced liver injury severity can vary significantly based on the time of administration, with more toxicity observed during the inactive phase in research animals [21]. The co-administration of artesunate and gentamicin may pose a risk of hepatotoxicity that could be amplified because of overlapping mechanisms involving oxidative stress and mitochondrial dysfunction, as the two drugs induce the formation of ROS, initiate a signaling cascade of inflammation, and disrupt mitochondrial respiration. This study provides chronopharmacological insights into time-of-day and seasonal modulation of hepatotoxicity, contributing to the understanding of chronotherapeutic optimization in malaria-endemic settings.
METHODS
Chemicals and reagents
Gentamicin (150 mg ampoule; Lek Pharmaceuticals) and sodium artesunate (120 mg vial; Lincoln Pharmaceutical; Batch No: EK4006; Expiry: Oct 2026) were used. Chloroform, ethanol, and formaldehyde were obtained from Sigma-Aldrich. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) kits were purchased from Abcam (ALT: ab105134; AST: ab105135; ALP: ab83368). All reagents were of analytical grade.
Drug preparation and dosing
Artesunate was administered at 100 mg/kg. For a 200 g rat, the required dose was 20 mg, which was prepared as a 50 mg/mL (reconstitute 120 mg with 2.4 mL sterile water). The injection volume for a 200 g rat was 0.40 mL, delivered intraperitoneally (IP).
Gentamicin was administered at 120 mg/kg, corresponding to 24 mg for a 200 g rat. For combination groups, volumes of each drug were halved and administered in separate syringes, with artesunate given immediately after gentamicin.
Animals and housing
Male Wistar rats (190–220 g) were obtained from the National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria. They were housed in the animal facility of the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, under ambient temperature conditions of approximately 26°C and a natural 12-hour light–dark cycle, with ad libitum access to standard chow and water. Animals were allowed to acclimatize for 3 weeks before the experiment.
Ethical considerations
All procedures complied with Directive 2010/63/EU and institutional guidelines. The study protocol was approved by the Ahmadu Bello University Animal Care and Use Committee (Approval no: ABU/CAUC/2024/024).
Experimental design
A total of 128 rats were initially enrolled and randomized into 16 groups (n=8 per group) across two seasons (rainy: August 2023; dry: February 2024) and two dosing times (12:00 h and 00:00 h). Treatments were administered once daily via intraperitoneal (IP) injection for 7 days. Four animals died before final sampling; thus, the final sample size used for analysis was 124.
During the rainy season, the first eight groups (Groups 1–8) received the following treatments:
• Group 1 (control): normal saline (1 mL/kg) at 12:00 h
• Group 2: gentamicin (120 mg/kg) at 12:00 h
• Group 3: artesunate (100 mg/kg) at 12:00 h
• Group 4: gentamicin (120 mg/kg) + artesunate (100 mg/kg) at 12:00 h
• Group 5 (control): normal saline (1 mL/kg) at 00:00 h
• Group 6: gentamicin (120 mg/kg) at 00:00 h
• Group 7: artesunate (100 mg/kg) at 00:00 h
• Group 8: gentamicin (120 mg/kg) + artesunate (100 mg/kg) at 00:00 h
The same experimental design was repeated during the dry season (Groups 9–16). In both seasons, all treatments were administered once daily by the IP route for 7 consecutive days. For the group that received the drug combination, the volume of each drug was reduced by half (in separate syringes with artesunate administered immediately after gentamicin was administered) to ensure that all rats received the same total volume of injection.
Randomization, blinding, and environmental conditions
Animals were randomly assigned to treatment groups using a randomization schedule to ensure equal distribution across seasons and dosing times. Four animals were excluded from the study because they died before the final day of treatment, and 124 rats completed the experimental protocol. Sample-size rationale was based on previous chronotoxicity studies in rodents using similar biochemical endpoints, where a minimum of 6–8 animals per group was sufficient to detect medium-to-large effect sizes with a power of ≥80%. A sample size of 8 per group provides 80% power to detect a 25% change in ALT with α=0.05.
Environmental parameters for each season were recorded: during the rainy season (August 2023), ambient temperature ranged from 22°C–27°C with high humidity (65%–78%). During the dry season (February 2024), temperatures fluctuated between 32°C–38°C with humidity between 18%–25%. Night-time dosing at 00:00 h was conducted under dim red light (<5 lux) to avoid circadian disruption, and animal handling procedures were standardized across seasons to minimize confounding influences.
Euthanasia and sample collection
On day 8, animals were euthanized by intramuscular ketamine (100 mg/kg). Blood (6–7 mL) was collected via jugular venipuncture, allowed to clot, centrifuged, and the serum was stored at -20°C until assay. Livers were excised and fixed in 10% neutral buffered formalin for histology.
Biochemical assay
ALT and AST were measured using kinetic enzymatic assays that monitor NADH oxidation at 340 nm (ALT: pyruvate + LDH; AST: oxaloacetate + MDH), with kits from Abcam (ALT: ab105134; AST: ab105135). ALP activity was determined using a kinetic colorimetric assay with p-nitrophenyl phosphate as the substrate, measuring absorbance at 405 nm (Abcam ab83368). The superoxide dismutase (SOD) activity was assessed by the Sun and Zigman method, based on the reduction of NBT measured at 560 nm. Catalase (CAT) activity was determined using the Aebi method by measuring the rate of H2O2 decomposition at 240 nm. Serum glutathione (GSH) levels were measured using a modified Tietze recycling method, monitoring the formation rate of TNB at 412 nm.
Determination of serum ALT
The process involves collecting blood and separating serum through centrifugation. Subsequently, ALT catalyzes the reversible transamination between L-alanine and α-ketoglutarate to form pyruvate and L-glutamate. The pyruvate formed is then reduced by lactate dehydrogenase (LDH) in the presence of NADH, which gets oxidized to NAD + . The decrease in absorbance due to NADH oxidation was measured spectrophotometrically at 340 nm.
Determination of serum AST
The serum is obtained from centrifuged blood. AST catalyzes the reversible transfer of an amino group from L-aspartate to α-ketoglutarate, forming oxaloacetate and L-glutamate. The oxaloacetate produced is then reduced to malate by malate dehydrogenase (MDH) using NADH, which oxidises to NAD + . The decrease in absorbance due to NADH oxidation is measured spectrophotometrically at 340 nm.
Determination of serum ALP
ALP catalyzes the hydrolysis of phosphate esters in an alkaline medium, liberating inorganic phosphate and an alcohol or coloured compound. The kinetic colourimetric method using p-nitrophenyl phosphate (pNPP) as the substrate is commonly employed. ALP hydrolyzes pNPP to release p-nitrophenol, which is colourless in acid but turns yellow in alkaline pH. The increase in absorbance at 405 nm (due to p-nitrophenol) is directly proportional to ALP activity.
Determination of SOD
The SOD activity was measured using the Sun and Zigman method [17]. The assay mixture contained 50 mmol/L potassium phosphate buffer (pH 7.8), 0.1 mmol/L EDTA, 9.9 mmol/L L-methionine, 5.7×10-5 mol/L NBT, and 2.5×10-2% Triton X-100. After adding the sample to the mixture, riboflavin was added to initiate the reaction, and the reduction of NBT was measured at 560 nm.
Determination of CAT
The CAT assay was performed using Aebi’s method. A phosphate buffer (50 mmol/L, pH 7.0) and 30 mmol/L H2O2 were used as reagents. The absorbance of the sample was measured at 240 nm and then again after 1 minute to determine CAT activity.
Determination of GSH
Serum GSH was quantified using a modified Tietze recycling method. Serum was deproteinized with 5% metaphosphoric acid, centrifuged, and the supernatant was neutralized. Total GSH was determined from the rate of TNB formation at 412 nm in a reaction containing DTNB, NADPH, and GSH reductase, and concentrations were interpolated from a GSH standard curve.
Histology
Liver tissues processed through graded alcohols, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin (H&E). Sections were examined at ×250 for necrosis, inflammatory infiltration, sinusoidal dilation, and central vein congestion.
Statistical analysis
Analyses were performed with GraphPad Prism v8.0.2. Normality was assessed with the Shapiro–Wilk test, and homogeneity of variances with Levene’s test. Two-way ANOVA (factors: season× dosing time) or three-way ANOVA (including treatment) was used as appropriate, followed by Bonferroni post hoc comparisons. Results are reported as F values, degrees of freedom, p values, partial η2, and 95% confidence intervals. Cohen’s d was calculated for key pairwise comparisons. Statistical significance was set at p≤0.05. The liver was carefully excised and fixed in 10% formalin solution for histological examination, and the findings are presented below.
RESULTS
Serum ALT
The results showed a significant seasonal difference (p<0.0001) in serum ALT levels in male Wistar rats. Rats treated during the dry season consistently exhibited higher ALT levels, indicating more significant liver toxicity than those treated during the rainy season. Significant differences were observed across various treatment times and combinations, with dry season treatments (gentamicin alone and gentamicin combined with artesunate) being more toxic. A more pronounced diurnal variation in hepatotoxicity was observed, with increased toxicity occurring during the dark phase compared to the light phase. Furthermore, the addition of artesunate to gentamicin during the dark phase resulted in a reduction of serum ALT levels in the rainy season. Notably, additive toxicity was evident during the dry season, where serum ALT levels were significantly elevated, as illustrated in Figure 1.
Determination of serum AST
The results showed a significant seasonal difference (p<0.05) in serum AST levels among the experimental rats. Treatments during the dry season consistently resulted in higher toxicity compared to the rainy season. Specifically, rats treated with gentamicin or gentamicin plus artesunate during the dry season had significantly higher serum AST levels than those treated during the rainy season (Figure 2).
Determination of serum ALP
The results showed a significant seasonal difference (p<0.0001) in serum ALP levels in the rats. Treatments during the dry season led to significantly higher ALP levels, indicating greater liver toxicity compared to the rainy season. Significant differences were observed across various treatment groups and times, including comparisons between gentamicin alone and gentamicin combined with artesunate, with dry season treatments consistently more toxic. While adding artesunate to gentamicin reduces liver toxicity during the rainy season, additive toxicity was observed during the dry season at the dark phase (p=0.0001) (Figure 3).
Serum SOD
Rats treated with gentamicin at 12:00 h during the rainy season showed significantly different (p<0.0001) SOD levels compared to other groups. Treatment at 12:00 h was more toxic than at 00:00 h, and dry season treatments were generally more harmful than rainy season treatments, especially when gentamicin was combined with artesunate (Figure 4).
Serum CAT
Serum CAT levels showed significant differences (p≤0.05) between rats treated with gentamicin at 12:00 h during the rainy season and those treated with gentamicin or gentamicin-artesunate combinations during the dry season, highlighting enhanced oxidative damage in the dry season groups (Figure 5).
Serum GSH peroxidase
Rats treated with gentamicin at 12:00 h (rainy season) significantly (p<0.0001) differed in GSH levels from those treated with gentamicin–artesunate at 00:00 h (rainy and dry seasons). Significant differences were also observed between rainy season and dry season treatments, with dry season exposure generally showing more significant toxicity. Gentamicin-only groups differed significantly (p=0.006) across seasons (Figure 6).
Histological observations of the liver
Photomicrographs revealed that rats treated with normal saline at 12:00 h and 00:00 h showed relatively normal liver histo-architecture in the rainy or wet season. In contrast, gentamicin plus artesunate treatment caused notable histo-architectural distortions, including cellular infiltration, dilated sinusoids, and congested central veins. Combined gentamicin and artesunate treatment exacerbated these distortions, particularly at 00:00 h in the dry season (Figure 7).
The results of the ANOVA are summarized in Table 1.
DISCUSSION
The present study clearly demonstrates that hepatotoxicity induced by artesunate–gentamicin is jointly regulated by seasonal context and dosing time, with the strongest toxic responses occurring during the dry season and following nighttime (00:00 h) administration. This aligns with our enzyme, antioxidant, and histological findings, all of which consistently showed amplified injury under dry-season night dosing. These results correct earlier inconsistencies and clarify that the primary driver of enhanced toxicity is the interaction between seasonally elevated oxidative stress and circadian vulnerability during the inactive phase in rats. The results indicated significant diurnal and circannual variation in the hepatotoxicity of artesunate and gentamicin co-administration in male Wistar rats. In Nigeria, the northern region is mainly categorized into rainy and dry seasons. High temperatures and very low humidity characterize the dry season. Previous studies indicated that the temperature goes beyond 35°C during the dry season, with humidity dropping to less than 25%. For example, studies in the states of Kano and Katsina recorded a mean annual temperature of 33.4°C and 33.8°C, respectively [22]. Another study focused on Maiduguri and Yola, all in the north, observed that the evaporative cooling effect was from March to May, which is in line with the hottest months [23]. During the rainy season, August normally has the highest rainfall, the weather is the coolest, and the humidity is also high [24]. This study indicated that more toxicity was observed during the dry season than in the rainy season. This could be because environmental stressors such as high ambient temperature and low humidity can raise systemic oxidative stress [25]. This process causes the increased production of ROS, which in turn overwhelms the hepatic defense system, such as GSH, CAT, and SOD [26]. Another possible reason why more hepatotoxicity was observed in the dry season could be the upregulation of heat shock proteins because of increased temperature during the dry season [27]. Another study on perennial ryegrass looked into transcriptional responses of HSP and genes of anti-oxidant under combined heat and drought stress. It was discovered that genes such as HSP70, HSP90-6, and HSP26.2 were quickly upregulated, interestingly under combined stress conditions, suggesting a coordinated response to environmental challenges during the dry season [28]. This could sensitize the hepatocytes to toxicants such as artesunate and gentamicin by the alteration of protein stability and folding, making the liver more susceptible to drug-induced toxicity. A dry condition makes it possible to prime the liver toward a pro-inflammatory state, like TNF-α. IL-6 eventually increases hepatocellular injury when exposed to drugs like artesunate and gentamicin [29]. Previous work showed artesunate inhibits TNF-α-induced pro-inflammatory cytokine production by suppressing the NF-κB and PI3K/Akt signaling pathway in human rheumatoid arthritis fibroblast-like synoviocytes. This suggests that artesunate can modulate inflammatory responses, which influence its hepatotoxicity potential in a primed liver [30].
Additive toxicity was observed during the dark phase of the dry season because both drugs independently generate ROS and cause the depletion of mitochondrial function. The dry season is considered oxidative stress-heavy and could overwhelm the liver defense system, leading to lipid peroxidation, DNA damage, and protein oxidation. Artesunate is known to induce sterile inflammation through the release of damage-associated molecular patterns [31], while gentamicin could cause more inflammation by activating a Toll-like receptor on Kupffer cells [32]. These two combined effects could provide a synergistic inflammatory response that may amplify the hepatotoxicity observed in the dry season’s dark phase.
Gentamicin causes toxicity by inducing mitochondrial dysfunction, which leads to impaired ATP production and a rise in ROS levels [33]. High temperatures from the dry season could exacerbate mitochondrial permeability transition pore opening, which can cause necrosis and apoptosis [34]. Artesunate, an anti-malaria, acts by generating lethal free radicals in the Plasmodium parasites. When used with gentamicin under oxidative stress-prone dry season, additive toxicity is possible by enhanced lipid peroxidation and hepatocyte membrane disruption, as shown in Figures 1 to 4. A previous study by Habashy et al. [35] in 2019 showed that heat stress affects cellular antioxidant enzyme activity and oxidative stress biomarkers. GSH conjugation could be less effective in the dry season because of the depletion of GSH, which will reduce the detoxification capacity of hepatocytes, hence promoting artesunate and gentamicin-induced reactive metabolite toxicity.
The liver’s ability to perform metabolic activity follows a circadian rhythm pattern. Liver enzymes such as CYP450 activity are more active during the dark phase than the light phase because rats are nocturnal animals [36]. During the light phase, reduced metabolic activity could lead to the build-up of toxic metabolites that could be injurious to the liver. The mitochondria activity also follows a circadian rhythm pattern, with lower activity observed during the light phase; this leads to a high accumulation of ROS and thus increases vulnerability to artesunate and gentamicin.
Artesunate provided the hepatic-protected effect of gentamicin during the rainy season, as shown in Figures 1 to 4, where the addition of artesunate decreased serum liver enzymes. This could be possible because liver anti-oxidant properties may be higher during the rainy season. Artesunate at lower doses and at a lower oxidative stress level induces antioxidant pathways like Nrf2 activation, which causes the upregulation of cytoprotective genes [37]. The histology results support the finding of diurnal variation in hepatotoxicity following co-administration of artesunate and gentamicin in male Wistar rats, where distortion of the cellular integrity of hepatocytes was more pronounced during the light phase than the dark phase in the rainy season, but the reverse was the case in the dry season. More liver injuries were seen during the dry season than the rainy season.
In conclusion, both dosing time and season significantly influence the hepatotoxicity of artesunate–gentamicin co-administration in male Wistar rats. The most severe injury occurred following nighttime dosing during the dry season, indicated by enzyme increases, antioxidant depletion, and histological damage. Chronopharmacological factors should be considered when optimizing dosing schedules, particularly in malaria-endemic regions with strong seasonal variation.
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